Comparing the results obtained with Gram-negative and Gram-positive bacteria, Stat-1 activation in macrophages does not seem to be related to LPS content. The p-tyr701Stat-1 expression levels were found to be independent of the internalized bacterial number and IFN-γ release. On the contrary, Jak/Stat-1 pathway activation only occurs when an active infection has been established in the host macrophage, and it is plausible that the differences in the expression levels of p-tyr701Stat-1 could be due to different survival mechanisms or to differences in bacteria life cycles within macrophages.The infection of human macrophages by pathogenic bacteria induces different signaling pathways depending on the type of cellular receptors involved in the microorganism entry and on their mechanism(s) of survival and replication in the host cell. It was reported that Stat proteins play an important role in this process. In the present study, we investigate the changes in Stat-1 activation (phosphorylation in p-tyr Intracellular bacterial pathogens are a group of microorganisms that have developed many abilities to survive and replicate in mammalian cells ; thus, tAlthough any type of tissue cells potentially serves as a habitat, for most intracellular bacteria, the macrophage represents the typical host cell. Indeed, although macrophages contribute to the first line of defense against infection through phagocytosis, paradoxically, in host-pathogen interactions, they can also become a reservoir for several pathogenic bacteria. This occurs because these pathogens have evolved to reside within the hostile environment of macrophages and are able to avoid host cell death.When macrophages make contact with bacteria, several signal-transduction pathways are activated  stimulatThe signal transducer and activator of transcription 1 (Stat-1) is an indispensable component of the cellular response to interferons (IFNs) during the immune reaction to pathogens. In fact, interferons employ receptor-associated Janus kinases (Jaks) to activate Stats by tyrosine phosphorylation . Mycobacterium avium infection induces Stat-1 hyperactivation for a considerable period and that it is important for macrophage survival after the establishment of an infection. Hence, it represents a potential target for drugs lowering phosphorylate Stat-1 expression levels.Recent findings suggest that Stat proteins play an important role in bacterial infections and in the activation of the innate immune response . In fact M. avium or inert particles induced a fast and transient activity of the Jak/Stat-1 pathway when internalized by Fcγ receptor engagement, probably as a preventive defense mechanism. However, a persistent Stat-1 pathway activation occurs only when an intracellular M. avium infection is established in macrophages.In a subsequent study  we showeBacteria can be classified into two major groups, Gram-positive and Gram-negative, and their classification depends on staining determined by different cell wall components. In Gram-negative bacteria there is a strong presence of lipopolysaccharides (LPSs). LPSs are a potent macrophage-activating stimulus inducing the expression of several genes necessary for the execution of host defense function .Listeria monocytogenes, Staphylococcus aureus) and Gram-negative  species responsible for infectious diseases in humans.Interestingly, LPS and IFN are known to induce expression of a common set of genes in sensitive cell types such as macrophages. Although Stat-1 appears to play an essential role in various forms of innate immunity, its role in LPS-inducible gene expression is not fully understood . Thus, sL. monocytogenes, a Gram-positive bacterium causing infections in immune-compromised patients and pregnant women  and 1 mM PMSF (phenyl-methylsulphonylfluoride), and phosphatase inhibitors: 1 mM sodium orthovanadate and 1 mM of sodium fluoride). Cells lysates were boiled immediately for 5 min and centrifuged at 6,000 ×g for 13 min. The protein concentration of cell extracts was determined by the Lowry assay . For theGermany) , 27. For, Italy) . Quantit post hoc test with the Graph Pad Prism 5 software. The significance of the data was evaluated on the control cells. A P value of less than 0.05 was determined to be statistically significant.The results are presented as means ± standard error (SEM) of at least 3 separate experiments. Statistical analyses were performed by the ANOVA type ES. aureus and L. monocytogenes) and two species of Gram-negative (S. typhimurium and L. pneumophila) intracellular bacteria to evaluate Stat-1 signaling pathway modulation after the phagocytosis of these pathogens by human macrophages. Macrophage cells were infected as reported in In this study we selected two species of Gram-positive ( S. typhimurium increased up to 1 log and declined slightly (0.5 logs) after 72 hours from phagocytosis. In contrast, intracellular S. aureus, L. monocytogenes, and L. pneumophila decreased in a time-dependent manner after phagocytosis ( S. aureus after 24 (up to −1.91 logs) and 72 (up to −3.87 logs) hours and for L. pneumophila after 72 hours (up to −3.81 logs), while the viable L. monocytogenes declined more gradually: up to −0.41 and −2.18 logs after 24 and 72 hours, respectively. Taken together these results confirm the differences among the four bacteria species in terms of their ability to survive intracellular killing in macrophages.As shown in ocytosis . In part S. aureus, L. monocytogenes, S. typhimurium, and L. pneumophila. Then after 0, 24, and 72 hours, the levels of p-tyr701Stat-1 expression, used as a marker of Stat-1 pathway activity, were correlated with the amount of pathogens present inside the macrophage cells. In Figures 701Stat-1 expression levels do not always decrease in parallel with the number of bacteria in the cells.In one set of experiments macrophages were infected with701Stat-1 expression levels increase immediately after phagocytosis reaching values up to 600% and 70% higher than values measured in uninfected cells for S. aureus . At 24 hours the increment of p-tyr701Stat-1 persisted, with values up to 135% higher than controls, while at 72 hours after infection, levels were once again found to be 300% higher than controls. In L. pneumophila infected macrophages, p-tyr701Stat-1 expression levels increased most significantly after 24 hours after phagocytosis (up to 80%), and in this case as well, these values were measured when pathogens were present in the host cell. Decreased levels of Stat-1 phosphorylation correlated with the elimination of the bacteria from the host.Gram-negative bacteria showed a similar trend to Gram-positive bacteria.  Figures  and L. p Figures . S. typh701Stat-1 pathway activity is independent of INFγ release. In fact, macrophages infected with both L. monocytogenes and S. typhimurium showed high levels of expression of p-tyr701Stat-1 also in the presence of an antibody blocking INFγ release. The increased p-tyr701Stat-1 expression levels are consistent with the different ratio of replication of the two pathogens, respectively.Data reported in Intracellular pathogens are responsible for much of the worldwide morbidity and mortality caused by infectious diseases. These pathogens are so dangerous because of their ability to survive and grow inside host macrophages . Phagocy M. avium infection in macrophages kept the Jak/Stat-1 pathway active for a considerable period (more than 7 days), subsequent to the establishment of an intracellular infection and bacteria replication in the phagosome [Various findings suggest that Stat proteins play an important role in bacterial infections , 32. In hagosome , 34.701Stat-1 overexpression gives the infected cells a perfect target for cytotoxic drugs, such as fludarabine, which exert their action by inhibiting Stat-1 transcription [We have previously postulated that p-tyrcription . It has cription .L. monocytogenes and S. aureus) and two Gram-negative bacteria (S. typhimurium and L. pneumophila) responsible for infectious diseases in humans.In the present study we investigated Stat-1 activation after the uptake of four bacterial species with varying abilities to enter, survive, and replicate in human macrophages. For this purpose we selected two Gram-positive bacteria  molecule, the major component of the outer membrane of Gram-negative bacteria, activates the Jak/Stat-1 pathway through LPS-induced CD40 expression . In our se IFN-γ , 37, whi701Stat-1 after blocking the release of INFγ suggest that internalization and replication of pathogens in macrophages potentially activates the Stat-1 pathway by intracellular signaling. The exceptionally high expression levels of p-tyr701Stat-1 reconfirms Stat1 pathway activation as a marker to identify bacterial reservoirs. Such reservoirs could be eliminated after treatment with drugs blocking the Stat-1 pathway of Stat-1 phosphorylation. Further experiments will be carried out using animal disease models.Taken together, the findings of this work show a strong activation of the Stat-1 pathway in different intracellular pathogens without significant differences between Gram-positive and Gram-negative bacterial pathogens. The unchanged expression levels of p-tyr


Background. Poor inpatient glycaemic control has a prevalence exceeding 30% and results in increased length of stay and higher rates of hospital complications and inpatient mortality. The aim of this study was to improve inpatient glycaemic control by developing an alert system to process point-of-care blood glucose (POC-BG) results. Methods. Microsoft Excel Macros were developed for the processing of daily glucometry data downloaded from the Cobas IT database. Alerts were generated according to ward location for any value less than 4 mmol/L (hypoglycaemia) or greater than 15 mmol/L (moderate-severe hyperglycaemia). The Diabetes Team provided a weekday consult service for patients flagged on the daily reports. This system was implemented for a 60-day period. Results. There was a statistically significant 20% reduction in the percentage of hyperglycaemic patient-day weighted values >15 mmol/L compared to the preimplementation period without a significant change in the percentage of hypoglycaemic values. The time-to-next-reading after a dysglycaemic POC-BG result was reduced by 14% and the time-to-normalization of a dysglycaemic result was reduced from 10.2 hours to 8.4 hours. Conclusion. The alert system reduced the percentage of hyperglycaemic patient-day weighted glucose values and the time-to-normalization of blood glucose. Hyperglycaemia and hypoglycaemia are prevalent in the inpatient setting. A recent analysis of hospital glucometry data by Bersoux and Cook from January to December 2012 was conducted on 51.4 million measurements from 2.6 million inpatients. The authors found that the prevalence of hypoglycaemia (<4 mmol/L or 70 mg/dL) was 6.1% in non-ICU patients and 5.6% in ICU patients, while the prevalence of hyperglycaemia (>10 mmol/L or 180 mg/dL) was substantially higher at 32% in non-ICU patients and 28% in ICU patients. The mean point-of-care blood glucose (POC-BG) was 167 mg/dL for non-ICU patients and 170 mg/dL for ICU patients. The authors concluded that increased hospital participation in data collection was needed for the development of optimal practices to manage inpatient dysglycaemia [p < 0.01) [Numerous studies have shown that improvement in glycemic control results in lower rates of hospital complications in general medicine and surgery patients –6. In on < 0.01) .At the opposite end of the glycaemic scale, studies have shown that in-hospital secondary hypoglycaemia increases inpatient mortality, likelihood of readmission, and length of stay –9.Certain clinical situations increase the risk of dysglycaemia during a hospital admission. These include changes in caloric or carbohydrate intake especially “nil by mouth” status or total parenteral nutrition ; use of There is evidence that poor inpatient glycaemic control is underrecognized, underreported, and suboptimally managed and that proactive assessment of inpatients' glycaemic status and aggressive treatment approaches are required –19. The Current networked point-of-care technology offers a novel way to address the problem of inpatient dysglycaemia and clinical inertia.http://dx.doi.org/10.1155/2015/807310). 3.4% of readings were in the hypoglycaemic range and 10% of readings were in either moderate or severe hyperglycaemic ranges. On average 12 patients per day had POC-BG results in the moderate-severe hyperglycaemic range, with more than 4 times that number having values >10 mmol/L. The mean time-to-next POC-BG test in a patient with a dysglycaemic value was almost 6 hours with a range of 2 minutes to 33 hours and the mean time-to-normalization of blood glucose after a dysglycaemic result was approximately 10 hours with a range of 1 to 76 hours.Our hospital conducted an audit of inpatient glucometry results from our Accu-Chek Inform II  meters aWe aimed to develop an alert system where all POC-BG measurements within the moderate-severe hyperglycaemic and hypoglycaemic ranges would be reviewed and notified to the relevant ward teams, nursing staff, and Diabetes Consult service. The metric chosen to analyse glucometry data was the patient-day model, which has been shown to most faithfully reflect the quality of inpatient glycemic control . We aimeThe glucometers used in this study were the Accu-Chek Inform II system  . All hoshttps://app.box.com/s/kk0ka29wmuwk855hdbq4yfqvh7nshfnp).Microsoft Excel version 2013  and Visual Basic version 13  were used to develop Excel Macros for processing of glucometry results from the Adelaide and Meath Hospital, Dublin, Ireland. An explanation of the Visual Basic Code for the Macros is available in the Supplementary Appendix. A sample anonymised data file and a working version of the Macro-Enabled Workbook is also available online , normoglycaemia (4–10 mmol/L), mild hyperglycaemia (10–15 mmol/L), moderate hyperglycaemia (15–20 mmol/L), and severe hyperglycaemia (>20 mmol/L). Alerts were generated for any hypoglycaemic result and for any result in the moderate-severe hyperglycaemia range.This study was a 60-day prospective study on the impact of a glucometry alert system on hospital dysglycaemia, started on 11 April 2014. There was no Diabetes Registrar rostered to the project for a 14-day period from 05 May 2014 to 18 May 2014; care proceeded as per the preimplementation phase during this period.Approval was granted by the hospital Diabetes Consult team, hospital management board, laboratory manager, and information technology department. This project did not require submission to the ethics committee since it was aiming to improve the use of routine clinical data.Our Diabetes Consult service requested that the cutoff for review of a patient be 15 mmol/L rather than the ADA/AACE recommendation of 12 mmol/L since their capacity for reviewing patients was limited to 12–15 patients per day. There are many cutoffs used for classifying a glucose result as hypoglycaemic. Based on audit data showing that only 3.4% of our hospital's results were less than 4 mmol/L, the cutoff for hypoglycaemic alerts was set at this level. The touchscreen tablet was used by the Diabetes Consult Registrar to highlight the hospital patients with poor glycaemic control. The Consult Registrar reviewed the patient's history, hospital stay, and insulin regimen. The alert system was used on weekdays only since there was no Consult service on weekends.Please review this patient's in-hospital glycaemic control. We have noted episodes of hypoglycaemia (<4 mmol/L) and/or hyperglycaemia (>15 mmol/L) on the point-of-care glucometry alert system. Refer to the hospital guidelines on glycaemic management and/or request a consult from the Hospital Diabetes Team.The point-of-care manager, Chemical Pathology trainee, or another member of the biochemistry staff performed daily uploads of dysglycaemic results to WinPath , our laboratory information system. A Glucose Alert (GA) was added to WinPath for every dysglycaemic result with the following comment:The glucose alert uploads to the LIS were performed only on weekdays during the study period.Data downloaded from the Cobas IT 1000 database were analysed using Stata version 13 . POC-BG results were analysed for the pre- and postimplementation periods, from 10 February 2014 to 10 April 2014 and from 11 April 2014 to 9 June 2014, respectively. Results from the acute care wards, emergency department, and pediatrics and outpatient wards were omitted because these have a substantially different patient demographic and/or are not representative of inpatient glycaemic control.t-tests were done to compare mean percentages of hypoglycaemia, hyperglycaemia, hypoglycaemic patient-days, and hyperglycaemic patient-days pre- and postpractice change. The daily mean patient-day weighted POC-BG values were displayed using line graphs with Lowess  lines to show trends over time.Summary statistics were obtained for the number of POC-BG results every day and the number and percentage of results <4 mmol/L and >15 mmol/L. The percentage of patient-day weighted mean blood glucoses <4 mmol/L and >15 mmol/L was calculated. Unequal variance one-sided t-tests, histograms, and normal density plots. The proportions of time values greater than 6 hours and 12 hours (time-to-next-reading) or greater than 12 hours and 24 hours  were also compared.For any POC-BG value <4 mmol/L or >15 mmol/L, the time-to-next-reading and the time-to-normalization of glucose were calculated. If the time between measurements was found to be greater than 72 hours, the patient was assumed to have been either discharged, admitted to ITU, or had serum blood glucoses analysed in the laboratory or on a blood gas analyzer; the time-to-next-reading was taken to be 72 hours in these cases. Comparisons of pre- and postpractice change were made using unequal variance one-sided Line graphs of the number of POC-BG tests performed per day and the percentage of mean patient-day weighted POC-BG values >15 mmol/L were plotted to investigate trends in glycaemic control and glucometry use over weekends.The Health Information Technical Standards of the Irish Health Information and Quality Authority (HIQA) were reviewed and followed closely to ensure data security and patient confidentiality . The HeaIn total, 45929 POC-BG results were downloaded from the Cobas IT database for the 120-day analysis period. After removing results from outpatient, paediatric and emergency departments, a total of 13992 results were analyzed for the preimplementation period compared to 14249 in the postimplementation period. There was no statistically significant difference between the numbers of POC-BG values, patient-day values, or patients for the two periods see .In total, there were 2023 dysglycaemic values and 272 patients reviewed by the Diabetes Consult Registrar after implementation. This corresponded to approximately 4 new patients and 10 known patients (from consults or from prior review) per day.p = 0.123). There was a 22.6% reduction in the percentage of patient-day weighted POC-BG values >15 mmol/L (p < 0.001), as shown in The frequency distributions of POC-BG values and mean patient-day POC-BG values by glycaemic level for the pre- and postimplementation phases are shown in p = 0.103) and in the percentage of patient-day weighted POC-BG values >15 mmol/L from 5.3% to 4.0%  with no change in the percentage of hypoglycaemic values and hypoglycaemic patient-day weighted values (p = 0.309 and p = 0.176). Furthermore, there was a greater reduction in the mean hospital patient-day weighted POC-BG from 8.22 mmol/L to 8.06 mmol/L (p < 0.01).A supplementary analysis was performed after omitting the 14-day period from 05 May to 18 May when no Registrar cover was available to review patients flagged by the alert system. After correcting for this protocol deviation, there was a reduction in POC-BG values >15 mmol/L from 11.2% to 10.4% (p < 0.01), as shown in p < 0.01). The density histogram shows a reduction in values over 6 hours, with a shift of the probability density plot to the left (see p < 0.001). The mean time-to-normalization of a dysglycaemic POC-BG result decreased by 19% from 10.2 hours to 8.3 hours (p < 0.01). The density histogram shows a fall in the number of values greater than 12 hours for this parameter compared to preimplementation. As shown in p < 0.05).There was a significant 14% reduction in the mean time-to-next-reading, from 5.1 hours to 4.4 hours (left see . 25.2% op = 0.008), highlighted by the red bars on The average number of POC-BG tests performed per day was approximately 230. There was a clear trend towards a lower number of tests on weekends with 240 ± 32 tests performed on weekdays compared to 223 ± 33 tests performed on weekends  issued guidelines in March 2010 recommending ketone (beta-hydroxybutyrate) measurement in the management of DKA . Ketone To determine whether there is a reduction in hospital cost, length of stay, and improved outcomes associated with improved glycaemic control due to the alert system, a six-month to one-year study is needed, with comparison to the previous year. Key metrics like length of stay and inpatient mortality, as well as perioperative outcomes for patients with a primary or secondary diagnosis of diabetes, will need to be recorded rigorously. The longer timeframe of the study will negate the effect of artefactual fluctuations in glycaemic control that are caused by the number of diabetics in hospital and other factors.An automated alert system that allowed the use of site-specific level and time-based criteria for the identification of dysglycaemic results and generation of alerts was successfully developed and deployed in our medium-large sized tertiary care facility. There was a significant improvement in our hospital's glycaemic control, with a reduction in the proportion of hyperglycaemic patient-days and no rebound increase in secondary hypoglycaemia. The alert system also improved the frequency with which glucometry tests were performed in patients with recognized out-of-control glucose results and improved the time-to-normalization of dysglycaemic results. The percentage of patient-day weighted results >15 mmol/L was reduced by greater than 20% and while the reduction in the time-to-normalization of dysglycaemic POC-BG results fell short of our 20% target, we successfully reduced the proportion of values greater than 12 hours by more than 20%. Certain trends in glucometry testing and glycaemic control were also observed, with lower numbers of tests performed and spikes in hyperglycaemia on weekends. Longer-term studies will enable us to determine whether improvements in glycaemic control correlate with improvements in in-hospital morbidity and mortality and reductions in length of stay and hospital costs.Supplementary Appendix 1 describes the methodology and results of a 5-day audit of inpatient glucometry results from our hospital's Accu-Chek Inform II glucose meters in October, 2012 to determine the rate of hypoglycaemia and hyperglycaemia in our hospital.Supplementary Appendix 2 provides a detailed description of the Excel Macros that were developed with sample illustrations of how data is processed using the Macros.


The aim of the present study was to construct tissue-engineered bone using a bioreactor and platelet-rich plasma (PRP). Bone marrow mesenchymal stem cells (BMSCs) and β-tricalcium phosphate (β-TCP) were cultured in a perfusion bioreactor with PRP-containing medium for 21 days to form a BMSC-TCP composite. Rabbits were then implanted with the BMSC-TCP composite. The morphology of the implanted BMSC-TCP composite was observed three months after surgery by scanning electron microscopy and hematoxylin and eosin (H&E) staining. In addition, the expression of cluster of differentiation (CD)31 and von Willebrand factor (WF) in the implanted BMSC-TCP composite was detected using immunohistochemistry. Bone formation was determined by comprehensive testing Following culture in a perfusion bioreactor and PRP, the BMSCs adhered to the β-TCP scaffold and the secretion of extracellular matrix was observed. The spreading and proliferation of cells was found to be enhanced on the scaffold. Furthermore, the vascular endothelial cell markers CD31 and VEF, were positively expressed. Therefore, these results suggest that tissue-engineered bone may be constructed using a bioreactor and PRP. PRP, which contains multiple growth factors, may promote vascularization of tissue-engineered bone. Avascular necrosis of the femoral head is usually caused by a lack of osteoblast progenitor cells and blood vessels . The forIn the present study, platelet-rich plasma (PRP) was used as a source of growth factors. PRP is an autologous platelet concentrate and is obtained from fresh whole blood by centrifugation. The biological activity of PRP depends on the enrichment in platelets. It has been confirmed that PRP prepared by appropriate technology contains a variety of growth factors at high concentrations, including platelet-derived growth factor, transforming growth factor-β, vascular endothelial growth factor, insulin-like growth factor and epidermal growth factor. PRP has an important role in promoting the proliferation of bone precursor cells in various animals, including rabbits and goats ,5. In adet al . β-TCP was obtained from Shanghai Bio-lu Biomaterials Co., Ltd. . Hematoxylin and eosin (H&E), anti-cluster of differentiation (CD)31 antibody, anti-von Willebrand factor (vWF) antibody and 3,3′-diaminobenzidine (DAB) chromogenic reagent for immunohistochemistry were purchased from Boster Biological Technology Co., Ltd. . Low-glucose Dulbecco’s modified Eagle’s medium (DMEM) was obtained from HyClone .The perfusion bioreactor used in this study was jointly designed by our group and the East China University of Science and Technology . The whole system consisted of a MasterFlex peristaltic pump , kcal head, perfusion column , air filters, two silicone tubes, a three-way connector and cell culture flasks. Two silicone tubes were placed into the cell culture flask. One silicone tube was immersed in the culture medium in order to extract the culture medium. The tube was then connected to the perfusion column to transport the medium. The culture medium flowed back into the flask through another silicon tube. The JSM-T300 scanning microscope was obtained from JEOL-Technics Co.  and the light microscope was from Olympus Optical Co., Ltd. .Ten male New Zealand rabbits, 2 months old, with a mean weight of 3.50±0.30 kg, were obtained from the Experimental Animal Center of Shandong Province . They were kept under standard conditions with free access to food and water.All animal experiments were approved by Animal Care and Use Committee of Shandong University and were conducted according to the ethical guidelines of Shandong University .The rabbits were anesthetized and 5 ml blood was collected from the ear vein. The blood was mixed with 1 ml sodium citrate anticoagulant. Then, the mixture was centrifuged at 549 × g for 6 min. Following centrifugation, the mixture was separated into a supernatant and a red blood cell layer, with an interface between them. The supernatant and the red blood cells 1–2 mm below the interface were transferred to another centrifuge tube and were centrifuged again . The supernatant was discarded and the remaining solution (~0.8 ml) was PRP. A total of 0.8 ml PRP was then mixed with 0.2 ml anticoagulant  and incubated for 10 sec in order to form a gel. The prepared PRP gel was then added to 25 ml culture medium to prepare PRP gel culture medium.6 cells/ml. The β-TCP scaffold was immersed in the cell suspension and the scaffold was then turned upside down to allow the cells to enter into the pores of the scaffold. The β-TCP scaffold was then cultured at 37°C in an incubator at 5% CO2. After 1 day, the BMSC-TCP composite (β-TCP scaffold covered with cells) was placed into the perfusion bioreactor column, with all the interfaces sealed by silicone. The entire system was then placed into an incubator at 37°C containing 5% CO2 and cultured for 21 days. The flow rate was maintained at 3 ml/min. By measuring the content of glucose in the culture medium, the culture medium was replaced every 2–3 days. The BMSC-TCP composite was examined for pathological morphology and used for further animal experiments.Rabbit BMSCs were cultured in low-glucose DMEM to the third generation and then the cells were adjusted to a single cell suspension of 5xl0Each rabbit was implanted with β-TCP and four different combinations of BMSCs and β-TCP, each at a different site. The five implanted materials were as follows: group A, β-TCP only; group B, uncultured BMSCs and β-TCP; group C, bioreactor-cultured BMSCs and β-TCP; group D, PRP-cultured BMSCs and β-TCP; and group E, bioreactor- and PRP-cultured BMSCs and β-TCP.The New Zealand white rabbits were anesthetized and placed in a left lateral position to fix their head and limbs. The right side was longitudinally incised to expose the superficial fascia. The BMSC-TCP composite was implanted between the subcutaneous superficial fascia and deep fascia. The position was recorded and the skin was sutured. The New Zealand rabbits were then kept under standard conditions and intramuscularly injected with 800,000 units penicillin each day, for 3 days following the surgery. The daily diet and wound healing were observed following the surgery.Three months following surgery, the BMSC-TCP composite was removed, and rinsed with saline. Then, the BMSC-TCP composite was fixed in 10% formalin, embedded in paraffin and cut into tissue sections. The tissue sections were dewaxed in xylene and rehydrated in graded alcohols. Following washing with running water and distilled water, the sections were stained with hematoxylin. After washing again, the sections were differentiated. Then, the sections were stained with eosin after washing with running water. Following dehydration and differentiation in alcohol, the sections were mounted and observed under a JSM-T300 scanning electron microscope.The expression levels of CD31 and vWF in the BMSC-TCP composites were detected using immunohistochemistry. Briefly, samples were fixed in formaldehyde and embedded in paraffin. Sections were dewaxed, rehydrated in graded alcohols and processed, prior to incubation with antibodies. After blocking, the sections were incubated with primary antibodies against CD13 and vWF. The sections were washed with phosphate-buffered saline, secondary antibodies  were then added and the sections were further incubated. The sections were then developed using the DAB chromogenic reagent and sections were counterstained with hematoxylin. Positive staining was presented as brown granules in cells. Five fields were randomly selected at high magnification under the light microscope and the number of positively stained cells was counted. The percentage of positive cells was the ratio of the number of positive cells to the number of total cells (≥26 cells were counted).The scoring criteria were as follows. Scores based on the percentage of positive cells: 0, percentage of positive cells ≤25%; 1, percentage of positive cells between 25 and 50%; 2, percentage of positive cells between 50 and 75%; and 3, percentage of positive cells ≥75%. Scores based on intensity of staining: 0, no positive staining; 1, weak  staining; 2, medium (brown) staining; and 3, strong (tan) staining. The histological score was the sum of the above two types of score, and a histological score of 0 was expressed as negative, a score 2 or 3 as weak positive, a score 4 or 5 as medium positive and score 6 or 7 as strongly positive.The SPSS statistical software package, version 17.0 was used for statistical analyses . Data are presented as the mean ± standard deviation. Analysis of variance was performed to compare differences between the different groups. P<0.05 was considered to indicate a statistically significant difference.Composite materials were implanted between the superficial fascia and deep fascia under the skin. For implantation, each rabbit was implanted at 5 different sites with β-TCP only (group A), BMSCs and β-TCP (group B), bioreactor-cultured BMSCs and β-TCP (group C), PRP-cultured BMSCs and β-TCP (group D), and bioreactor- and PRP-cultured BMSCs and β-TCP (group E), respectively. Following implantation, the wounds all healed, without any swelling, effusion and signs of infection. Three months following implantation, the BMSC-TCP composites were removed. To observe the morphology of the BMSC-TCP composites-, scanning electron microscopy and H&E staining was performed. Representative scanning electron microscopy results are shown in Representative H&E staining results for the various groups are shown in CD31 and vWF are expressed in vascular endothelial cells. Therefore, to evaluate the vascularization in the BMSC-TCP composite, immunohistochemical staining of CD31 and vWF was performed. Representative immunohistochemical staining results for CD31 and vWF are shown in in vivo cell stress environment. Therefore, the mechanical characteristics of the cells are similar to the biological changes that occur in vivo (In the present study, BMSCs were three-dimensionally cultured in a perfusion bioreactor. This culture model has certain advantages compared with traditional planar two-dimensional culture models. Firstly, the culture medium in traditional two-dimensional cell culture models requires intermittent replacement, whilst in a three-dimensional bioreactor the culture medium flows, and therefore does not need to be replaced frequently. However, the flow medium may stimulate cells mechanically and simulate an  in vivo –16. In a in vivo ,18. FurtIn our previous study , BMSCs w


Nasonia vitripennis is an emerging model organism that exhibits a strong photoperiodic response: Short autumnal days experienced by females lead to the induction of developmental arrest (diapause) in their progeny, allowing winter survival of the larvae. How female Nasonia control the developmental trajectory of their offspring is unclear. Here, we took advantage of the recent discovery that DNA methylation is pervasive in Nasonia and tested its role in photoperiodism. We used reduced representation bisulfite sequencing (RRBS) to profile DNA methylation in adult female wasps subjected to different photoperiods and identified substantial differential methylation at the single base level. We also show that knocking down DNA methyltransferase 1a (Dnmt1a), Dnmt3, or blocking DNA methylation pharmacologically, largely disrupts the photoperiodic diapause response of the wasps. To our knowledge, this is the first example for a role of DNA methylation in insect photoperiodic timing.Many organisms monitor the annual change in day length and use this information for the timing of their seasonal response. However, the molecular mechanisms underlying photoperiodic timing are largely unknown. The wasp  Seasonal timing is a key survival process for many organisms, especially in temperate regions. In most cases, light is the primary cue, and the seasonal change in day length is monitored by a so-called “photoperiodic clock” . DespiteNasonia is a typical temperate-zone insect, in which short photoperiods experienced by females during autumn induce developmental arrest (diapause) in the progeny at the larval stage . In a recent survey of methylation in Nasonia  gene has recently been shown to be critical for measuring photoperiod  pharmacological knockdown using the methylation inhibitor 5-aza-2′-deoxycytidine.Here, we test whether a similar encoding of environmental cues by DNA methylation underpins seasonal timing in insects. First, we provide a detailed genomic description of methylation in ata sets . Second,Nasonia vitripennis wasps kept in either long- or short-day conditions. We used reduced representation bisulfite sequencing (RRBS)   to achieNasonia reference genome was 84.1% (LD) and 83.2% (SD). The average coverage depth of the reads was 92.6× for the short-day sample and 86.4× for the long-day sample. By the same metric, 4.53%  of CpG sites in the genome were covered by both samples at an average depth of 95.72× for the SD sample and 88.97× for the LD sample. The LD reads covered 1,304,087 CpG sites , whereas the SD reads covered 1,385,067 CpG (4.94%). We detected 1618 and 2018 methyl CpGs in the LD and SD samples, respectively, using a 1% false discovery rate (FDR) (see Methods), comprising 0.124% and 0.146% of the cytosines with sequence coverage that were analyzed.After removal of low-quality reads, we obtained 27,255,357 reads (1.06 Gbp) from the long-day sample (LD) and 30,818,609 reads (1.25 Gbp) from the short-day sample (SD). The mapping efficiency of these reads to the Although most of the methylated cytosines were in a CpG context A, a smalNasonia  terms only to 25 genes in our list. The terms assigned to the genes represented a wide range of functions, such as GTPase activity, protein binding, and transferase activity (acyl and glycosyl groups). A few of these genes harbored multiple DMCs, including heixuedian (heix), an ubiA prenyltransferase domain containing protein 1 gene. Of the 42 CpG sites in this gene that were covered by our library, six were significantly more methylated in the short-day sample (misshapen (msn), encoding a protein kinase, was represented by seven CpGs sites in our reads, five of which showed increased methylation in the long-day sample (at the FDR-corrected q < 0.01) , LOC100117390 , and LOC100117821 (zinc finger protein 615) using a qPCR approach (P < 0.0001) .Of the 12,055 genes with annotated mRNAs in the Nvit_2.0 build, 5972 (49.54%) had at least five covered CpG sites, and 5032 (41.74%) had at least 10 covered CpG sites. We used Fisher's exact tests to compare methylation in long and short photoperiods and identified 51 differentially methylated CpG sites  , which wy sample B. In con < 0.01) C. We havapproach . In all Nasonia genome and identified a single putative Tet ortholog. Although the overall level of identity to human TET2 is rather low (0.25), the 925-bp Tet motif (Pfam ID: Tet_JBP) in the Nasonia gene results in a highly significant blast score (E = 4.9 × 10−60), suggesting it is a bona fide Tet ortholog. We carried out hydroxymethylated DNA immunoprecipitation (hMeDIP), using Nasonia gDNA that was spiked with mouse DNA  compared to the positive control. Thus, functional 5-hMC might exist in Nasonia but is unlikely to play a significant role in the photoperiodic response. The complete survey of 5-hMC in Nasonia will be published elsewhere.During bisulfite conversion, 5-hmC has been shown to behave like its precursor, 5-mC . We haveouse DNA , and tesDnmt1a-c and Dnmt3 by injecting double-stranded RNA (dsRNAi) of these genes into female pupae of the wild-type N. vitripennis strain AsymC. Control females were injected with dsRNAi targeting the green fluorescent protein (GFP). We verified the knockdowns by qPCR of mRNA extracted from the injected females . Although the complete knockdown of Dnmt1a is lethal , the response of Dnmt1a-dsRNAi-injected females was similar in both long and short days , with a substantial increase in diapause-induction in progeny of females maintained under long-day conditions. Silencing Dnmt3 . This effect of the dsRNAi was observed within 10 d of injection. For progeny laid 10–15 d after injection, diapause frequencies reverted to normal levels again . We have also attempted the knockdown of Dnmt1b and Dnmt1c, but the progeny of the injected wasps showed normal photoperiodic response .To test the functional role of DNA methylation in mediating short-day-induced diapause, we knocked down s lethal , our prong Dnmt3 B resulteCG11148 (LOC100117390), WDR36 (CG9799), and LOC100117821. Knockdown of Dnmt1 resulted in a significant reduction of methylation  in both the WDR36 and LOC100117821 loci, whereas for Dnmt3 knockdown, the level of methylation was reduced in the treated group (DNMTs-dsRNAi) compared to the control group (GFP-dsRNAi) in each of the tested genes (P < 0.05).We verified the effect of the RNAi knockdown on CpG methylation by using qPCR (MethyQuant), 9–10 d post injection (5 d after eclosion) We analyzed the status of highly methylated CpG sites in three different genes : P < 0.001) (P = 0.47). Intriguingly, in this case, we observed a reduction of diapause in the progeny of wasps maintained in short day (median drop from 35% to 25%), accompanied by an increase in long-day progeny (8% to 20%). We verified the impact of the drug on DNA methylation of CpG sites in four different genes , LOC100118618 (heix), LOC100679542 , and LOC100122664 . A mixed effects model ANOVA  revealed a significant reduction in methylation due to the drug  = 9.45, P = 0.015). All genes exhibited the same response  = 0.74, P = 0.56). We also tested the effect of 5-aza-dC on msn by cloning a 335-bp fragment of the gene using DNA from either treated or control females. Comparing the overall methylation in four CpG sites that were identified by RRBS revealed a significant reduction in the drug-treated group  compared to the control group (n = 9 clones): one methylated and 39 unmethylated sites compared to six methylated and 30 unmethylated sites (Fisher's exact test P = 0.048).We also tested the impact of DNA methylation pharmacologically. We used the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza-dC), a potent inhibitor of DNA methylation , and tes< 0.001) A, wasps nt genes B: LOC100Nasonia vitripennis. Patterns of methylation across the genome change with photoperiod, and manipulation of the methylome via either the disruption of a key maintenance methyltransferase (Dnmt1a) or via a potent inhibitor of methylation (5-aza-dC) changes how female Nasonia respond to photoperiod. In addition, we have obtained a single-nucleotide resolution snapshot of the methylome of Nasonia that adds to the number of recently published insect methylomes, including the honeybee Apis mellifera  because there are other physiological processes that might change with day length . Here, our goal has been to demonstrate that differential methylation with respect to photoperiod is present . Yet, some of the DMCs in our list may serve as candidates to follow. For instance, the gene eceptors  and therDnmt1a demonstrates the causative role of DNA methylation in the photoperiodic response (Dnmt1a RNAi). As such, the differences in response in the two experiments may reflect multiple pathways and targets of DNA methylation with various effects on diapause, which are likely to be locus- and tissue-specific. Consistent with the fact that the change in day length is associated with both hyper- and hypomethylation  during early embryogenesis led to developmental arrest and lethality. It is also possible that differences between the Dnmts are caused by their noncatalytic activity. Such noncatalytic regulation of gene expression has been demonstrated in the vertebrate DNMT1  agents. Likewise, in honeybee caste determination, the methylation level in the brains of workers and queen shows both up- and down-regulation in a gene-specific manner , which cIn summary, our experiments clearly demonstrate a significant role of DNA methylation in photoperiodic timing. How these methylation changes underlie the maternal transfer of the photoperiodic response to the progeny remains to be determined. The fact that DNA methylation is involved in the photoperiodic clock echoes the role of epigenetic regulation in the circadian clock . Indeed,Nasonia  at 18°C under various photoperiods (using LED illumination) for 10 d. Hosts were replaced twice, after 5 and 9 d, and kept in darkness at the same temperature for an additional 20 d. At this stage hosts, were dissected and diapause status of the progeny was determined (diapause larvae are easily identified through their morphology).We used the wild-type strain, AsymC, whose genome has been sequenced . To testNewly eclosed females were maintained in either a long (18 h) or short (6 h) photoperiod. After 10 d, 2 h after lights on, genomic DNA was isolated from 10 females using the DNeasy kit (Qiagen). The RRBS libraries were prepared as previously described . Brieflyhttp://www.bioinformatics.babraham.ac.uk/projects/fastqc) was used to provide data quality information at each step of the quality control process. The data were taken through several preprocessing steps before being aligned to the Nasonia genome (version Nvit_2.0) using Bismark v.0.7.7 (http://hannonlab.cshl.edu/fastx_toolkit/), which was also used to remove potential noise . To facilitate downstream analysis, the resulting Bismark SAM output was sorted by chromosome and position using SAMtools version 0.1.18 . The raw sequencing data was obtained through two Illumina sequencing runs on the same libraries, yielding 117,646,358 raw 50-bp reads in total. FastQC version 0.10.1  due to incomplete bisulfite conversion and sequencing error is given by E = nCf/(nCf + nTf), where nCf is the number of Cs found at the filled-in position; and nTf is the number of Ts found at the filled-in position. The error estimates for each sample were 0.0068 (LD) and 0.00622 (SD).P-values that had been generated in this way for each site were adjusted using the Benjamini-Hochberg method (To classify a cytosine as methylated (mC), the binomial distribution was used to calculate the probability of getting K successes (methylated Cs) in N trials (read depth) with probability E of a success . The P-vg method , settingP-values were corrected to genomic-wide false discovery rate (FDR)-based Q-values by using the SLIM method . Information about the tested genomic regions and primer sequences are in Supplemental Table S2.To test for 5-hydroxymethylation, we used the previously published hmeDIP protocol . Brieflyn = 2000) of randomly reallocated versus non-reallocated data  (Nasonia transcriptome (Nvit_2.0) and its complement.The protocol for generation of escribed . For thelas.com) . The tooAequeorin mRNA (a jellyfish photoreceptor) was spiked to the RNA samples and served as an exogenous reference  mixed with 10 µM of the drug (without a host). After 2 d, each female was given two hosts and transferred to 18°C at either a long- or short-day photoperiod. The hosts were collected after 5 d, and progeny were inspected after 20 d as above.Following injection, surviving females were collected and kept in different photoperiods as above. qPCR (SYBR green-based) was carried to determine transcript levels of the targeted gene, using the standard curve method. eference , and thehttp://www.ncbi.nlm.nih.gov/geo/) under accession number GSE44869. The DNA methylation data can also be browsed at WaspAtlas (http://waspAtlas.com), our new Nasonia genomic database (All high-throughput sequencing data from this study have been submitted to the NCBI Gene Expression Omnibus (GEO; database .


Psoriasis is a chronic inflammatory skin disease and topical therapy remains a key role for treatment. The aim of this study is to evaluate the influence of psoriasis-like lesions on the cutaneous permeation of anti-psoriatic drugs.We first set up imiquimod-induced dermatitis in mice that closely resembles human psoriasis lesions. The development of the lesions is based on the IL-23/IL17A axis for phenotypical and histological characteristics. Four drugs, 5-aminolevulinic acid (ALA), tacrolimus, calcipotriol, and retinoic acid, were used to evaluate percutaneous absorption.2/h by imiquimod intervention. However, imiquimod did not promote skin deposition of this macrolide. The lipophilicity, but not the molecular size, dominated drug permeation enhancement by psoriatic lesions. The in vivo percutaneous absorption of ALA and rhodamine B examined by confocal microscopy confirmed the deficient resistance of epidermal barrier for facilitating cutaneous delivery of drugs via psoriasis-like skin.The most hydrophilic molecule, ALA, revealed the greatest enhancement on skin absorption after imiquimod treatment. Imiquimod increased the skin deposition and flux of ALA by 5.6 to 14.4-fold, respectively, compared to normal skin. The follicular accumulation of ALA was also increased 3.8-fold. The extremely lipophilic drug retinoic acid showed a 1.7- and 3.8-fold increase in skin deposition and flux, respectively. Tacrolimus flux was enhanced from 2 to 21 μg/cmWe established the topical delivery profiles of anti-psoriatic drugs via imiquimod-treated psoriasis-like skin. Psoriasis is a chronic inflammatory skin disease characterized with epidermal hyperplasis, leukocyte infiltration, inflammation, and increased vascularity in dermis . It is wDrug permeation by topical products is generally examined via normal or intact skin; however, topical products are usually applied to diseased skin. Some efforts have been done previously for comparing the drug penetration between intact and damaged skins. The approaches for producing damaged skin for permeation evaluation include heating, delipidization, tape stripping, abrasion, and photoaging –6. The iWe employed a mouse model to mimic the aspects of psoriasis. Topical application of imiquimod, a ligand of toll-like receptor (TLR) 7 and 8, has been recently reported to induce psoriasis-like dermatitis . Four drALA, retinoic acid, and rhodamine B were purchased from Sigma-Aldrich . Tacrolimus was a standard reference from U.S. Pharmacopeia . Calcipotriol was supplied by Cayman . The 5% imiquimod cream (Aldara) was a gift from 3M Pharmaceuticals .Female BALB/cByJNarl mice at 7 weeks old were supplied by National Laboratory Animal Center . This study was carried out in strict accordance with the recommendations in the Guidelines for the Care and Use of Laboratory Animals of Chang Gung University of Science and Technology. Ethical issues with animal experiments complied with Directive 86/109/EEC from the European Commission. The protocol was approved by the Institutional Animal Care and Use Committee of Chang Gung University of Science and Technology (Permit Number: 2013–005). The alfalfa-free food  and water were given ad libitum. All efforts were made to minimize suffering.This method was modified from the protocol by van der Fits . The mouThe gross imaging of mouse skin treated with or without imiquimod cream was observed using a handheld digital microscope . A magnification of 200x was used for imaging capture.2/h). A spectrocolorimeter  was used to quantify cutaneous erythema. The pH was determined by Skin-pH-Meter PH905 (Courage and Khazaka).Immediately after an 8-day application of imiquimod, the mouse skin was examined for physiological parameters, including transepidermal water loss (TEWL), erythema, and skin surface pH. A Tewameter  was employed for measuring TEWL .The dorsal skin was excised from the mouse after sacrifice. The skin species were immersed in a 10% buffered formaldehyde using ethanol, embedded in paraffin wax, and sliced at a thickness of 3 μm. The samples were stained by hemoxylin and eosin (H&E) and imaged under light microscopy . For immunohistochemistry (IHC) observation, the biopsies were incubated for 1 h at room temperature with primary antibodies against the following cells or antigens: CD3g for 4 min. The supernatant was collected and the cytokine concentration was quantified by using Bio-Plex Cytokine Assay Kit  according to the method described previously [The skin samples were rinsed by cell wash buffer. A 500 mM phenylmethylsulfonyl fluoride in cell lysis buffer (0.5 ml) was prepared and added into test tubes with skin samples. The probe-type sonicator  was used to homogenize the mixture for 10 s. Subsequently the mixture was freezed by liquid nitrogen. After thawing at room temperature, the mixture was homogenized by the sonicator for 10 s again. The sample was then centrifuged at 4500 xeviously . The preTotal RNA was extracted with Trizol (Invitrogen) and cDNA was synthesized by reverse transcription using iScript cDNA Synthesis Kit (Bio-Red). The qRT-PCR was performed using iQ SYBR Green Supermix (Bio-Red) according to the manufacturer’s instructions. The housekeeping gene GAPDH was used as an internal control and gene expression was calculated using comparative the threshold cycle method. The following primers were used to analyze target mRNA expression: TNF-α: 5’-CCCTCACACTCAGATCATCTTCT-3’ (forward) and 5’-GCTACGACGTGGGCTACAG-3’ (reverse); IL-17A: 5’-GCTCCAGAAGGCCCTCAG-3’ (forward) and 5’-CTTTCCCTCCGCATTGAC-3’ (reverse); IL-23: 5’-CCCGTATCCAGTGTGAAGATG-3’ (forward) and 5’-CCCTTTGAAGATGTCAGAGTCA-3’ (reverse); filaggrin: 5’-GTTTCCAAACACATGGATCAAAT-3’ (forward) and 5’-TTTGAATCTTGTTGGTGTCTGTG-3’ (reverse); involucrin: 5’-AAACTTGGTGAGCCAGAATTACA-3’ (forward) and 5’-CCTTTCCAGTTGTTTACCCTTCT-3’ (reverse). GAPDH was an internal control: 5’-AGCTTGTCATCAACGGGAAG-3’ (forward) and 5’-TTTGATGTTAGTGGGGTCTCG-3’ (reverse).2. The receptor temperature and stirring rate of the stirrer in receptor were kept at 37°C and 600 rpm, respectively. At determined intervals, a 300-μl medium was withdrawn from the receptor. After a 24-h application, the skin was removed from the Franz cell. The permeant amount within the cutaneous reservoir was extracted by 0.1 N HCl for ALA and methanol for the other drugs.The experiment was conducted with Franz diffusion cell . The excised skin with or without imiquimod treatment was mounted between the donor and receptor compartments with SC facing upward into the donor. The receptor medium was pH 7.4 buffer (5.5 ml) for ALA to maintain a sink condition. The receptor contained 30% ethanol in pH 7.4 buffer for the permeation of tacrolimus, calcipotriol, and retinoic acid. The donor (0.5 ml) was loaded with ALA , tacrolimus , calcipotriol , or retinoic acid . The pH 5 buffer, 30% propylene glycol (PG)/pH 7.4 buffer, 30% ethanol/water, and 30% PG/water was employed as donor vehicle for ALA, tacrolimus, calcipotriol, and retinoic acid, respectively. The effective diffusion region for permeants was 0.785 cmAll samples were analyzed by high-performance liquid chromatography (HPLC) for estimating drug concentration in skin and receptor. The HPLC setup  for ALA, calcipotriol, and retinoic acid was described in our previous studies –14. For Differential stripping and cyanoacrylate skin surface casting were utilized to detect the content of drugs in hair follicles . Subsequ2 was attached to mouse back by superglue. An aliquot of 0.2 ml pH 5 buffer containing ALA (0.64%) or 30% PG/water containing rhodamine B (0.03%) as the permeant was pipette into the cylinder. The application period was 6 h. The animal was then sacrificed, and the treated skin area was excised. A confocal laser scanning microscopy  was used to observe horizontal section of the skin. The thickness of skin was scanned at ~5 μm increments via z-axis. Images were taken by summing 15 fragments at different depths from skin surface.A glass cylinder with a hollow area of 0.785 cmp < 0.05) was taken as the level of significance.Statistical analysis of differences between the groups was performed using Kruskal-Wallis test. The post hoc test for checking individual differences was Dunn’s test. A 0.05 level of probability (2/h. The imiquimod-treated group had a significantly higher (p < 0.05) level of TEWL (36.90 g/m2/h). TEWL is associated with the degree of barrier damage. There was a slight increase in erythema in the imiquimod-treated skin compared to control skin, however, this difference was not statistically significant (p > 0.05)  . In addi > 0.05) .p > 0.05)  the production of IL-1β, GM-CSF, and TNF-α by 2.5-, 2.3-, and 1.7-fold, respectively. Imiquimod intervention did not alter (p > 0.05) the levels of IL-12 and IFN-γ.The mediators of inflammation such as cytokines are important to govern psoriatic pathophysiology. We used the Bio-Plex technique to determine the cytokine levels in the control and imiquimod-treated skin. As shown in Some cytokines were also determined by the mRNA levels using qRT-PCR analysis as shown in P) of four permeants used for testing skin penetration into/across the control and imiquimod-treated skin. The cutaneous delivery of the drugs was assessed using the Franz diffusion assembly method. Both retention in the skin and the permeated amount across the skin were evaluated. The accumulation in the skin indicates cutaneous uptake by topical delivery, whereas the permeated amount predicts transdermal delivery to the systemic circulation. P of -1.5. The skin deposition of ALA in control skin was 0.18 μg/mg. The amount of the drug accumulated in the skin reservoir for the imiquimod-treated skin was 5.6-fold higher than the control skin. The flux across the psoriasis-like skin was 14.4-fold higher than the control skin. The follicular route, a primary pathway for drug delivery, is used to determine the effect of topical therapies on the appendageal pathway. The amount of ALA deposited in the follicles was evaluated by the combination of stripping and cyanoacrylate casting. The recovery of ALA from the casts is demonstrated in 2 of ALA was recovered from the follicles of the control skin. The ALA amount in the hair follicles was found to increase approximately four-fold after being treated with imiquimod.P = 3.3). It has a large molecular size with a MW of 822 Da. Tacrolimus levels in the skin between the groups were not significantly different (p > 0.05) (p > 0.05). Calcipotriol possesses a log P (3.8) similar to tarcolimus but has a lower MW (413 Da). In the imiquimod-treated skin the accumulation of calcipotriol was 0.24 μg/mg, which was significantly greater (p < 0.05) than that of the control skin  across skin compared to the control group. There was no relevant follicular uptake in the control group. Calcipotriol was found to accumulate preferentially in the follicles of imiquimod-treated skin (0.91 μg/cm2).Tacrolimus is an anti-psoriatic drug with a moderate lipophilicity (log  > 0.05) . NeverthP of 6.3. As shown in p < 0.05) for the imiquimod-treated group (0.34 μg/mg) compared to the control group (0.21 μg/mg). The flux across imiquimod-treated skin was 0.73 μg/cm2/h, which was approximately four times greater than that across the control group. The imiquimod-treated group had a retinoic acid uptake in the follicles of 1.75 μg/cm2, a four-fold increase over the control group.Retinoic acid belongs to the extremely lipophilic drug with a log P of 1.95, distributed in the mouse skin was detected following in vivo administration to the dorsal area for both groups. ALA or rhodamine B, a fluorescent dye with a log Differing from ALA distribution, rhodamine B distribution is constrained to the epidermal region of normal skin . Rhodami+ cells and a decrease in the expression of β-catenin and involucrin on imiquimod-treated skin. The IL-17A, IL-23, and TNF-α also revealed over-expression in this psoriasis-like skin model. It is recognized that the appearance and histology of imiquimod-treated skin was somewhat different from that of van der Fits [The mouse model was utilized in this study, as it has been useful in simulating psoriasis states . In thisder Fits . No erytder Fits , a fluct+ cells and CD11c+ dendritic cells [+ lymphocytes in mouse epidermis. Various types of cytokine are involved in the modulation of inflammatory responses in inflammatory skin diseases like psoriasis. Imiquimod has been reported to elicit IL-17A and IL-23 levels in mouse skin, which are features of human psoriasis [+ cell infiltration in psoriatic lesions. The previous report [+ cells in psoriasis upregulated the expression of IL-23. IL-23 induces several psoriasis features such as acanthosis and erythema [+ cells also promote GM-CSF production, stimulating the growth of inflammation-related granulocytes such as neutrophils and eosinophils [TEWL can be a reflection of skin-barrier function, including the constituents of SC and tight junctions. The experimental results showed an increased TEWL by imiquimod, indicating the disruption of the epidermal barrier by psoriasis. Activated T cells are thought to be the primary modulators of psoriatic pathogenesis. Psoriasis is infiltrated with immune cells, mainly CD3ic cells . Our IHCsoriasis . It actssoriasis . TNF-α psoriasis . This res report  has demoerythema . IL-23 ainophils .IL-17, IL-23 and GM-CSF are involved in the T-helper (Th)17 cell response . Th1 immIL-17 can raise the expression of antimicrobial peptides and decrease cell adhesion factors, thus impairing the skin barrier function . We furtAccording to the skin penetration profiles, most of the permeants could facilely diffuse into/across psoriasiform skin compared to healthy skin. This indicates that the barrier function defect, and not elongation of delivery pathlength, dominates drug absorption through psoriatic skin. The full epidermis, including SC and epidermal junctions, offers a permeability barrier for drug diffusion. Psoriasis-induced barrier impairment occurs via SC disruption according to TEWL increment. The barrier was also weakened by the loss of β-catenin and involucrin. These deficiencies are momentous factors in the enhancement of drug absorption via psoriatic skin.psoriasis/control of skin deposition in our study (5.6-fold). Topically-applied tacrolimus has been verified to show poor absorption and inefficient therapy in regard to psoriatic lesions due to the thick plaques [The SC is proved to be a critical permeation barrier for ALA . SC dama plaques ; our resCalcipotriol could locate within normal skin without any penetration into receptors after topical application. This result was consistent with clinical observation that calcipotriol has very low systemic absorption . The treP. The percutaneous absorption of more hydrophilic drugs would be more affected by the skin with compromised barrier function. Tight junctions can efficiently prevent the diffusion of permeants with a MW of > 600 Da [The steady-state flux of the permeants across skin can be influenced in terms of lipophilicity and molecular size . For the> 600 Da . Barrier> 600 Da . ImiquimThe follicular density in skin varies depending on the sites on the body, with the scalp holding the highest density . On the in vivo cutaneous distribution of ALA and rhodamine B. It can be seen in the CLSM image that the PpIX level in psoriasis-like epidermis was higher than that in normal epidermis; the facile ALA penetration into defective skin barrier was responsible for this increment. Another possibility is the reduced ferrochelatase activity and increased porphobilinogen deaminase by the hyperproliferative tissue [We further explored e tissue , which ee tissue , who sugBesides the therapeutic efficacy of psoriasis treatment modalities, an important concern is the drug tolerance and the safety of the patients. PDT with ALA causes skin edema and inflammation in some cases due to direct damage to fibroblasts and mast cells . The comAnother concern is that most of the investigations have employed normal skin for assessing percutaneous absorption of the drugs. This can underestimate the real absorption in the diseased skin. Some attempts such as penetration enhancers, iontophoresis, microneedles and nanocarriers are undertaken to enhance ALA flux for successful delivery into target sites . Normal Although a successful result was achieved in this report, it should be cautious to reflect the clinical condition by these data using the mouse as the animal model. The mouse skin is thinner and more-permeable as compared to human skin. Though the porcine skin is a well-recognized alternative for human skin , there iThis study demonstrated that imiquimod-induced psoriasis-like skin in mice is an effective model for investigating drug activity. Our findings demonstrated the increased skin deposition and flux of the drugs by psoriasis-like skin as compared to healthy skin. The drug lipophilicity was an essential factor controlling permeation enhancement by imiquimod. The most pronounced enhancement was observed with hydrophilic drugs. The imiquimod treatment did not largely alter skin accumulation and the flux of lipophilic permeants. We found that SC disturbance by imiquimod dramatically promoted cutaneous drug absorption. Most investigations have used intact skin for evaluating drug absorption. The results from such research may not be feasible for predicting cutaneous delivery of the drugs on diseased or damaged skin. The skin model used in this work is beneficial for resolving this issue, especially the anti-psoriatic activities.S1 Text(DOCX)Click here for additional data file.


Pseudomonas sp. strain MT-1 was the first deep-sea denitrifier isolated and characterized from mud recovered from a depth of 11,000 m in the Mariana Trench. We report here the genome sequence of this bacterium, which contributes to our understanding of denitrification and bioenergetics in the deep sea. Pseudomonas sp. strain MT-1 is a deep-sea denitrifier isolated from the Challenger Deep of the Mariana Trench . The suitable growth conditions for MT-1 are temperatures between 4 and 45°C , hydrostatic pressures between 0.1 and 50 MPa , NaCl concentrations between 0 and 10% , and pH 6 to 10 . From 16S rRNA gene analysis, MT-1 appears to be closely related to Pseudomonas stutzeri and Pseudomonas aeruginosa, but it was shown that P. stutzeri adapts to lower temperatures and higher hydrostatic pressures than does P. aeruginosa . Automatic annotation was performed using the Microbial Genome Annotation Pipeline (The genome sequence of Pipeline .nar), periplasmic nitrate reductase (nap), and nitrous oxide reductase (nos) were identified in the genome of MT-1 (–nir] and nitric oxide reductase [nor]) were identified. The nir gene was found to be of the nirS type, consistent with previous spectroscopic analysis. These were also similar to corresponding genes of the related pseudomonads.As described previously, the gene sets for membrane-bound nitrate reductase  were found. It is known that many anaerobic respiratory terminal enzymes, like nitrate reductase, contain molybdopterin as a cofactor. The existence of multiple molybdoenzymes suggests that Pseudomonas sp. strain MT-1 might utilize multiple compounds as a substrate in anaerobic respiration. These might support the growth of the bacterium in the deep-sea sediments.The annotation of the genome sequence showed that this bacterium has 22 genes encoding Pseudomonas sp. strain MT-1 was deposited at DDBJ/EMBL/GenBank under the accession no. AP014655. The version described in this paper is the first version, AP014655.1.The genome sequence of


Mycobacterium tuberculosis (MTB) with its L-forms (MTB-L) and lung cancer. In the present study, we have optimized and applied a highly sensitive assay to detect the presence of MTB and MTB-L in 187 lung cancer samples and 39 samples of other cancer origins. By carefully controlling confounding factors, we have found that 62% of the lung cancer samples are MTB-L positive, while only 5.1% of the other cancer samples are MTB-L positive. Through generalized linear models and random forest models, we have further identified a set of clinical end-points that are strongly associated with MTB-L presence. Our finding provides the basis for future studies to investigate the underlying mechanism linking MTB-L infection to lung cancer development.There is a recent emerging theory that suggests a cross-link between pathogens and cancer. In this context, we examined the association between the  Lung cancer is a leading cause of death with an annual mortality rate of 1.59 million people, accounting for 19.3% of all cancer mortality worldwide . Therefo Mycobacterium tuberculosis (MTB), primarily as a pathogen of the mammalian respiratory system, is closely linked to the occurrence of lung cancer [ Mycobacterium tuberculosis L forms (MTB-L) are a wall-defective and pleomorphic form of MTB and have many characteristics different from MTB's original vegetative forms [It has been speculated thatg cancer . Approxig cancer , with 1%g cancer . Of partg cancer . Mycobacve forms . While ive forms . mpb64 are used in in situ hybridization. Gene mpb64 is in the MTB genome, and its MPB64 protein is identified from MTB strain H37Rv [Generally, MTB-L have low pathogenicity and activity. Therefore, MTB-L have often gone undiagnosed and have not been identified as the cause of disease . For exain H37Rv , 9. Althin H37Rv , 11.Currently, there have been limited investigations on the relationship between lung cancer and MTB-L. Our lab is a key pioneer in this field and published two primary studies. In these two studies, we showed that MTB-L exist in lung cancer patients as the primary type of MTB. Unfortunately, we were unable to explain any of the tumorigenic mechanisms yet , 11. in situ hybridization using mpb64 probes. We compared the rate of MTB/MTB-L positive cases in lung cancer with the rate among other cancer types. By computational methods, we were able to extract key clinical features that can characterize a subpopulation of lung cancer samples with higher prevalence of MTB/MTB-L.In this study, we examined 187 lung cancer tissues for MTB/MTB-L presence by combining IK acid-fast staining andThe lung cancer samples were from 187 patients who were diagnosed with lung cancer at the North China Oilfield Hospital of Hebei Medical University. Here, they had surgery to remove the cancer from the lung between 2003 and 2012. A consensus cancer diagnosis was reached by pathology. For 131 out of 187 patients, complete pathological records were available. For 47 out of 131 patients, fresh surgery tissues were available. Within the 19 patients with history of TB, sixteen patients had ipsilateral tuberculosis and cancer, while another three had contralateral disease.For the TB, non-lung-cancer group, samples were from 31 patients who had surgery from the North China Oilfield Hospital of Hebei Medical University between 2004 and 2012 (14 out of 31 patients had fresh surgery tissues). Among the 31 patients, 19 had tuberculosis and 12 had tuberculous lymphadenitis.For the non-TB, non-lung-cancer control group, samples were from 39 hospitalized surgical patients in the North China Oilfield Hospital of Hebei Medical University between 2004 and 2012. Each patient went through a chest X-ray, CT, and OT tests that excluded the possibility of patients for having tuberculosis or lung cancer. 17 out of 39 patients had fresh surgery tissues. Among the 39 patients, 6 had hepatic carcinoma, 14 had gastric cancer, and 19 had breast cancer.The research protocol was approved by ethic committees of the North China Oilfield Hospital of Hebei Medical University where the samples were collected and all patients provided informed consent. All specimens were handled and made anonymous according to the ethical and legal guidelines. mpb64 gene  from the standard strain Mycobacterium tuberculosis H37R. The probes for the two fragments were made and biotin-labeled by Tianjin Haoyang Biotechnology Company  The probe sequences were as follows:5′-CGGTATCGGTGCCTTTCAACTCCTCGC-3′,5′-GGGCAGGCTGATGTTGATGTTGTAGGC-3′.Targeted fragments My250-224 and My310-283 were chosen from the in situ hybridization kit was purchased from Tianjin Haoyang Biotechnology Company. Other reagents such as 92-3TB, 92-3TBL, PNB-MTB and PNB-MTB-L, TCH-MTB and TCH-MTB-L, and other liquid media were prepared by our lab.The probes can be used separately or mixed together. In order to increase the MTB detection rate, these two probes were mixed for this experiment. The92-3TBL liquid media were made by first dissolving 2 g of glucose, 5 g of amino succinamic acid, 2.5 g of monopotassium phosphate, 1 g of monometallic sodium orthophosphate, 2.5 g of sodium citrate, 0.5 g of magnesium sulfate, 4 g of gelatin, 30 g of sodium chloride, and 20 mL of glycerol into distilled water to a final volume of 900 mL.  4% NaOH was added to the solution to adjust its pH to 7.0. 1 mL of 1% malachite green  that was added to the solution, which was then split into vaccine bottles with 90 mL in each bottle. Bottles were placed under eight pounds of pressure for 15 minutes. After cooling to room temperature, 10 mL of sterile blood plasma was added into each bottle. (Fresh blood plasma was kept under 56 degrees Celsius for one hour followed by 4 degrees Celsius overnight. Blood plasma was brought up the next day by centrifuging at 1500 ×g for 30 minutes.) Finally, aseptic technique was used to split the mixture into small test tubes with two to three mL in each tube. Samples were cultured in an incubator for 48 hours to ensure no bacterial contamination.92-3TB liquid media was made following the same procedure as that for 92-3TBL liquid media, with the exception that no sodium chloride was added.p-Nitrobenzoic acid- (PNB-) MTB or MTBL liquid media were made by adding PNB  into 92-3TB or 92-3TBL liquid media to make the final PNB concentration to 0.25 mg/mL.2-Thiophenecarboxylic acid hydrazide- (TCH-) MTB or MTBL liquid media were made by adding TCH  into 92-3TB or 92-3TBL liquid media to make the final TCH concentration to 0.25 mg/mL.μm.Slide preparation: tissue was harvested and fixed immediately with 4% paraformaldehyde  and 0.1 M PBS ), followed by dehydration, paraffin embedding, and sectioning of a series of 6 slides with a thickness ranging from 4 to 6 Intensified Kinyoun's (IK) acid-fast staining to detect MTB-L: as used here, this method has previously been described in the literature . BrieflyThe Ziehl-Neelsen staining to detect MTB-bacteria type: the slides were dewaxed and went through the following series of ethanol dehydration . First, carbol fuchsin solution was made by dissolving 4 g of Schiff stain  in 100 mL of 95% ethanol  to make 100 mL of saturated solution. Second, 10 mL of saturated solution was mixed with 90 mL of 5% phenol . Third, 3% muriatic acid ethanol solution was made by adding 3 mL of concentrated hydrochloric acid  into 97 mL of 95% ethanol . Lastly, Loffler's alkaline methylene blue solution was made by dissolving 2 g of methylene blue  into 100 mL of 95% ethanol  to make 100 mL of saturated solution. The final solution was made by taking 30 mL of saturated solution and mixing it with 100 mL of distilled water and 0.1 mL of 10% caustic potash . These methods strictly followed TB Diagnostic Bacteriology Testing Protocols developed by the China Tuberculosis Association.In situ hybridization to detect mpb64 gene expression: following the procedure from the kit manual, the probe was hybridized to a lung cancer tissue slice and used as the positive control, while a lung cancer tissue without probe hybridization was used as the negative control. The detection method was NBT/BCIP method. If the hybridized cancer cell nucleus showed brown particles, it was deemed mpb64 positive. To determine the mpb64 expression status per slice, five high magnification views (400x) from each slice were selected at random. A mpb64-positive-cancer-cell rate for each view was determined by counting the percentage of mpb64-positive cancer cells in all cancer cells under each view. The final positive rate for each slice was then calculated as an average of the rates from the five views. The overall sample's mpb64 expression was deemed positive if the per-slice positive rate is greater than or equal to 5%; otherwise, it was labeled as mpb64 negative.Use of bacterial culture to detect MTB and MTB-L: part of the tissue from each resecting surgery was placed onto a sterile plate, cut by sterile scissors, and ground. Each half gram of ground tissue was then inoculated into 92-3TB and 92-3TBL liquid media, mixed, and incubated at 37 degrees Celsius for one to three weeks. The sample was observed once each week by taking the precipitate onto slides for the IK acid-fast staining and the Ziehl-Neelsen staining before microscopy observation. Bacteria samples that were grown on 92-3TB liquid media were transferred to PNB-MTB and TCH-MTB liquid media to identify the MTB type. Bacteria samples that were grown on 92-3TBL liquid media were passed to the same media (92-3TBL) from generation to generation. Each generation was cultured for one week until the fifth generation. Meanwhile, samples were inoculated onto PNB-MTL-L and TCH-MTB-L liquid media. The identification method for MTB-L was the same as for MTB above.The response of the data was calculated with following equation: Samples with missing values were filtered out. The final dataset contained seven covariates and 99 samples , 69 of wr square was calculated for investigating goodness-of-fit. The training data was predicted by the model, and AUC was calculated as a measure of model accuracy. All statistics analyses were done with the R programming language [All covariates were put into an initial model. Next stepwise Akaike's Information Criterion (AIC) was applied to pick the strongest predictive features as well as to avoid overfitting. This procedure was repeated 1000 times, and the frequency of each variable appearing in the best model was used to determine the “important” features. Due to the unbalanced group sizes, in each iteration, we utilized the bootstrap resampling technique to generate 100 positive samples and 100 negative samples as the training data. Pseudo language .Random forest is an ensemble of classification trees that are calculated on random subsets of the data, using a subset of randomly restricted and selected predictors for each split in each classification tree. In this study, we used the “party” R package  to perfoOriginally, we sought to establish a reliable assay for detecting MTB-L by assessing and optimizing existing standard assays. The Ziehl-Neelsen (ZN) staining had traditionally been used as a standard method for detecting MTB  in TB samples. Recently, reports have leveraged the emergence of Intensified Kinyoun's (IK) acid-fast staining technique to detect MTB in general, and more specifically the L forms , 14.In the current report, we first assessed our ability to detect MTB-L by the IK method. Discriminatory detection of MTB-L by this method has a significant histology component (see Methods) and depends on the experience of the histologists . A sampl mpb64 gene in the cell nucleus, a marker for MTB [ mpb64 marker improved the true positives rate in TB samples for MTB/MTB-L from 93.5% to 100% while only slightly decreasing the true negatives rates in non-TB samples from 94.9% to 91.7%  contrast with the samples from other cancer types . Among other cancer types, there were one out of 14 gastric cancer samples (7.1%) and one out of six breast cancer samples (16.7%) detected as MTB-L positive, which was still a statistically significant contrast. To increase sensitivity  higher than 3 of 39 samples from other cancer types (7.7%). Among other cancer types, there were one out of 14 gastric cancer samples (7.1%) and two out of six breast cancer samples (33.3%) detected as MTB-L positive.A total of 187 lung cancer samples were collected for the present study. Of these samples, 19 patients had a documented prior medical history of TB and 113 had no reported TB history, while the remaining 55 patients were missing this information. We selected the 113 samples with no reported TB history to compare with 39 samples of other cancer types without prior TB medical history. As shown in sitivity , MTB/MTBP value = 0.016) lower than 61.9% in lung cancer. For MTB/MTB-L, 60 out or 115 samples (52.2%) were positive, which was still lower than 64.6% in lung cancer  [To investigate the relation between MTB and breast cancer, we conducted a breast cancer study. Out of 115 breast cancer patients, we found that 53 samples (46.1%) were MTB-L positive, which is statistically significantly , however, indicated that a linear model alone is likely insufficient, especially without explicit inclusion of critical interaction terms.We next applied a generalized linear regression model as a representative of linear models to examine potential combinatorial effects of all clinical end-points. The final model identified category and lymph node metastasis as having the strongest combined effects . The strWe then removed the assumption that the association is strictly linear and derived a random forest model as a representative of nonlinear models to reexamine the association. Again random forest model was run on a balanced dataset through bootstrapping. We repeated the procedure five times and the average AUC was 0.85 . We used a standard R package to run random forest model, which should be replicable. The relatively high rates of lung cancer and TB cases in China have recently sparked new theories that there might be cross-associations between the two diseases. Recent reports of colocalization of MTB/MTB-L in tumor samples, although limited in sample size and coincidental in nature, underline the need for thorough confirmation studies. Towards this goal, we set up the present study with three key components in mind: (1) accurate detection assay; (2) large sample size; and (3) full medical history and clinical diagnosis to control for confounding factors as well as linkage to clinical end-points.mpb64) for detection. In this study, we proved that the IK method is better than the ZN method, but the mpb64 in situ hybridization method can further increase sensitivity. By combining the IK method and the mpb64 in situ hybridization method, we could find out samples that had MTB but in a hard-to-stain form, which is worth future study.Acid-fast staining methods are the traditional way for MTB and MTB-L detection, and isolation is the gold standard for validation. However, we noticed that staining methods failed to detect MTB or MTB-L in some MTB-L positive samples that were validated by isolation. We hypothesized that there might be a group of MTB which is hard to be stained because of unknown characteristics. In this situation, because this group should still contain the main components of MTB, we could make use of an important MTB protein coding gene  or that of gastric cancer sample (7.1%); see This finding suggested that the presence of MTB-L might be strongly associated with lung cancer, but the causal relationship was underdetermined. Because MTB-L are wall-defective and pleomorphic, they have the potential to act like carcinogenic viruses. They can get into human alveolar epithelial cells, remain dormant, and induce cancer by DNA integration, because DNA integration can potentially activate oncogenes and/or repress tumor suppressor genes. In mouse model, researchers reported that MTB-L infected mouse had significant higher chance to develop neoplasia than the control group , 17. TheWhile the biological mechanism of causality is still unclear, we acknowledged that preclinical experiments beyond the scope of the present report were needed to fully investigate the question of causality. Nevertheless, we reasoned that a causal relationship would also reflect an association with clinical disease severity.Towards this goal, we focused on finding end-point differences between MTB-L positive and negative lung cancer samples. None of the individual end-points in isolation was sufficient to distinguish between the positive and negative samples. However, upon applying computational classification algorithms, we were able to identify a set of clinical end-points that could be used to stratify between the samples. To our knowledge, this was the first report linking clinical end-points to the presence and absence of MTB-L. As our rule-based model indicated, MTB-L was especially prevalent in patients with non-lymph-node-metastatic small-cell lung and squamous cell carcinoma. Our findings should lead to further studies focusing on this subpopulation, as well as making a strong case for the advantage of utilizing primary cells from this subpopulation as an experimental model system. A reliable detection assay for MTB/MTB-L was developed. MTB-L is more prevalent in lung cancer samples compared to other cancer types. A set of clinical end-points, which are strongly associated with MTB-L presence, was identified.